Identification of Novel Modulators of Opioid Receptor Signaling During Drug Addiction

Opiates are the most widely used analgesics for the treatment of pain; however, prolonged treatment with opioids leads to the development of tolerance. Genetic and pharmacological inhibition of PKC in rodents demonstrated that PKC is a key player in inducing morphine tolerance. Mu opioid receptors (MORs) contain more than 15 serine, threonine, and tyrosine residues that are accessible to protein kinases. We intend to characterize differential phosphorylation of MORs and other PKC substrates upon activation by morphine versus fentanyl. In addition, we intend to identify PKC targets leading to opioid tolerance using a combination of novel tools and modern proteomics approaches. Previous studies have suggested that protein kinase C (PKC) is a key player in inducing morphine tolerance and addiction. In order to investigate the dynamics of PKC activity in native tissue we generated antibodies to several substrates of PKC and used these in combination with conformational-sensitive antibodies that detect time- and ligand-mediated activation of native PKC and explored the dynamics of PKC activity. In cells treated with morphine or fentanyl, we find that PKC activation induced by morphine is rapid and transient whereas the activation by fentanyl is slow and sustained. In animals with morphine administration we detect robust activation of PKC in the striatum. Using the Center’s Discovery Proteomics Core we intend to identify the PKC phosphorylated sites on mu opioid receptor activated by morphine/fentanyl.

In a related project we find that prolyl endopeptidase-like enzyme (PREPL) plays an important role in opioid receptor endocytosis. Specifically, in cells with the knock-down of PREPL protein, we find a decrease in the rate and extent of ligand-induced receptor internalization. A critical question in the field has been the mechanism by which PREPL mediates this function. Towards this end, we would like to characterize the substrates of PREPL. We are currently evaluating PREPL’s enzyme activity and will use the Center’s expertise to characterize the substrates of PREPL.